Internal mobility along the mainchain of a number of small proteins has been monitored using 1H-detected 15N NMR relaxation experiments. Extending this approach to the proton bearing carbons would enhance the understanding of protein dynamics not only due to the much larger number of sites, but also by monitoring sidechain dynamics for which a wider range of internal mobility is anticipated. Unfortunately, such 13C relaxation experiments are complicated by two major impediments. Although isotope labeling is necessitated in all but the smallest protein systems, uniform enrichment gives rise to homonuclear scalar and dipolar interactions which confound the standard relaxation experiments. Furthermore, dipole-dipole interference effects severely complicate the interpretation of relaxation data of methylene and methyl resonances. Although partial solutions exist for the case of the symmetric methyl system, the methylene case remains problematic. Both of these impediments can be overcome by combining two isotopic labeling patterns previously developed in this laboratory, alternating carbon enrichment and random fractional deuteration. In the first labeling pattern each residue type is enriched with a 13C-12C-13C... pattern out along each sidechain thus providing high levels of enrichment without one bond 13C-13C couplings. Random fractional deuteration combined with multiplet editing sequences and 2H gated decoupling can yield suppression of signals from carbons bearing two protons so that the monoprotio signal can be obtained and analyzed analogous to the methine and mainchain amide resonances. Relaxation analysis will be carried out on E. coli thioredoxin and staphylococcal nuclease. Both proteins have reported high resolution x-ray structures as well as NMR assignments and structural studies. E. coli thioredoxin has been used to show the practicality of this labeling approach and initial studies qualitatively indicated the range of dynamics present. Quantitative dynamical analyses will be carried out. The larger staphylococal nuclease is the premiere model system for biophysical and genetic approaches to the study of protein folding. The dynamics of the apo vs. pTp inhibited enzyme as revealed by NMR should provide insight into how the rigidification induced by substrate analog binding propagates through the protein structure. Random fractional deuteration effectively suppresses spin diffusion effects in NOE distance estimates. Quantitative comparison of these NOE distances with the high resolution x-ray structure will provide a useful statistical basis for assessing the reliability of NMR constraints in determining both local and global aspects of protein structure. Use of statistical weighting of constraints will markedly clarify present ambiguities in defining and assessing target functions in the structure refinement process. It is anticipated that the availability of more accurate NOE distance constraints along with the related statistical base will help resolve residual ambiguities resulting from comparisons between solution and crystal structures as well as enhance the confidence in solution structures of systems lacking corresponding x-ray analyses.